Three-dimensional endothelial cell sprouting assay (3D-ECSA) exhibits differentiation of endothelial cells into sprouting structures inside a 3D matrix of collagen I. It is a screening tool to study endothelial cell behavior and identification of angiogenesis inhibitors. The shape and size of an EC spheroid (aggregation of ~ 750 cells) is important with respect to its growth performance in presence of angiogenic stimulators. Apparently, tubules formed on malformed spheroids lack homogeneity in terms of density and length. This requires segregation of well formed spheroids from malformed ones to obtain better performance metrics. We aim to develop and validate an automated imaging software analysis tool, as a part of a High-content High throughput screening (HC-HTS) assay platform, to exploit 3D-ECSA as a differential HTS assay. We present a solution using Circular Hough Transform to detect a nearly perfect spheroid as per its circular shape in a 2D image. This successfully enables us to differentiate and separate good spheroids from the malformed ones using automated test bench.